Title

The N-Terminal Α-Helix Of Pancreatic Phospholipase A2 Determines Productive-Mode Orientation Of The Enzyme At The Membrane Surface

Keywords

interfacial activation; membrane binding; orientation; polarized infrared spectroscopy

Abstract

Phospholipase A2 (PLA2) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA2 activity strongly increases upon binding to the surface of aggregated phospholipid. The N-terminal ∼ten residue α-helix of certain PLA2 isoforms plays important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA2 and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal α-helix and the rest of the protein in membrane binding of PLA2 and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA2. While the full-length PLA 2 binds to membranes with a defined orientation, an engineered PLA2 fragment ΔN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits ∼100-fold lower enzymatic activity compared to the full-length PLA 2, indicating the key role of the N terminus in PLA2 function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA2 and identification of its interfacial binding surface. Moreover, the full-length PLA2 demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the ΔN10 fragment is more rigid than the full-length PLA2 both in free and membrane-bound states. Our results suggest that the N-terminal α-helix supports the activation of PLA2 by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA2 at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme. © 2004 Elsevier Ltd. All rights reserved.

Publication Date

11-12-2004

Publication Title

Journal of Molecular Biology

Volume

344

Issue

1

Number of Pages

71-89

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1016/j.jmb.2004.09.034

Socpus ID

7044269246 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/7044269246

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